In previous experiments, we showed that the in vivo transcription of the colicin E1 gene was dependent on cyclic AMP in adenylate cyclase-defective mutant cells of Escherichia coli (Ebina, Y. and Nakazawa, A (1983) J. Biol. Chem. 258, 7072-7078). We now show that cyclic AMP and cyclic AMP receptor protein stimulated the in vitro transcription of the gene in the presence of spermidine. As determined in DNase I protection experiments, two binding sites for the complex of cyclic AMP and the receptor protein were identified about 60 base pairs (CRP-1) and 110 base pairs (CRP-2) upstream from the transcription initiation site of the colicin E1 gene. CRP-1 had a higher affinity for the complex than that of CRP-2. Substituting an unrelated DNA sequence for CRP-2 reduced the efficiency of in vitro stimulation of the gene by cyclic AMP and the receptor protein. These potential binding sites for the cyclic AMP-cyclic AMP receptor protein complex probably participate in the stimulation of the colicin E1 gene transcription.